厌氧菌Sunxiuqinia sp. SH-52外切型海藻酸裂解酶的异源表达及酶学特征
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摘要
【背景】目前已报道的海藻酸分解菌多数为好氧菌,未见有关厌氧菌的报道。从分离的海藻酸分解菌中表征的海藻酸裂解酶大多为内切型海藻酸裂解酶,外切型较少。【目的】研究来自厌氧海藻酸分解菌的海藻酸裂解酶基因,表征新型海藻酸裂解酶并阐明其酶学性质,为海藻酸裂解酶的多样性和微生物降解海藻酸机制提供理论依据。【方法】对来自厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4编码基因进行克隆,分析基因序列,构建重组质粒PGEX-4T-1-SHA-4并在大肠杆菌中实现异源表达,经纯化后对其酶学性质及降解特征进行研究。【结果】该酶在28°C、用0.1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)条件下诱导6 h达到最大表达量,纯化后酶的比活力达到21 U/mg。酶学性质分析表明SHA-4的最适温度为37°C;最适pH为7.5;对PolyMG (杂聚古罗糖醛酸-甘露糖醛酸嵌段)具有底物偏好性;Na+对该酶的活性具有抑制作用,而金属离子Cu~(2+)具有明显促进作用,使活性提高了约168%;SHA-4催化海藻酸的Km值为2.5 mg/mL,Vmax为8.7 mg/(mL·min);SHA-4为外切型海藻酸裂解酶,降解海藻酸终产物为单糖。【结论】异源表达了来自一株厌氧海藻酸分解菌Sunxiuqiniasp.SH-52的海藻酸裂解酶SHA-4,该酶是PL6家族中第一个对PolyMG有底物偏好性的外切型海藻酸裂解酶,而且活性较高,作为工具酶有很好的应用前景,为海藻酸降解机制的探索提供了新的线索。
[Background] Most alginolytic bacteria currently reported are aerobic bacteria and studies on anaerobic ones have not been reported yet. Characterization of alginate lyases isolated from alginolytic bacteria are mainly focused on the endo-type alginate lyases and rarely on exo-type ones. [Objective] To investigate the genes encoding alginate lyases isolated from anaerobic alginolytic bacteria, to characterize the novel alginate lyase, and to elucidate its enzymatic properties, providing a theoretical basis for the diversity of alginate lyases and the mechanism of microbial degradation of alginate. [Methods] The gene encoding SHA-4, an alginate lyase isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp.SH-52, was cloned and sequenced. Recombinant plasmid PGEX-4 T-1-SHA-4 was constructed and heterologously expressed in E. coli. The expressed enzyme was purified and the enzymatic and degradation characteristics were analyzed. [Results] Maximum expression was achieved when induced by0.1 mmol/L IPTG(Isopropyl-β-D-Thiogalactoside) at 28 °C for 6 hours and the specific activity of the purified enzyme was 21 U/mg. Characterization of the enzyme showed that the optimal condition for SHA-4 includes the temperature at 37 °C, pH at 7.5, and PolyMG(heteropolymeric MG blocks) as a preferred substrate. The activity of this enzyme was inhibited by Na+ and significantly elevated to about168% by Cu~(2+). Km and Vmax of SHA-4 when used to catalyze alginate were 2.5 mg/mL and8.7 mg/(mL·min), respectively. SHA-4 was an exo-type alginate lyase with monosaccharides as the final degradation products. [Conclusion] An alginate lyase SHA-4 isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52 is successfully expressed. Among other PL6 family members, SHA-4 is the first exo-type alginate lyase with PolyMG as a preferred substrate. Along with its relatively high enzymatic activity, SHA-4 may serve as a tool enzyme with promising application and provide new clues for further exploration of the mechanism of alginate degradation.
[Background] Most alginolytic bacteria currently reported are aerobic bacteria and studies on anaerobic ones have not been reported yet. Characterization of alginate lyases isolated from alginolytic bacteria are mainly focused on the endo-type alginate lyases and rarely on exo-type ones. [Objective] To investigate the genes encoding alginate lyases isolated from anaerobic alginolytic bacteria, to characterize the novel alginate lyase, and to elucidate its enzymatic properties, providing a theoretical basis for the diversity of alginate lyases and the mechanism of microbial degradation of alginate. [Methods] The gene encoding SHA-4, an alginate lyase isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp.SH-52, was cloned and sequenced. Recombinant plasmid PGEX-4 T-1-SHA-4 was constructed and heterologously expressed in E. coli. The expressed enzyme was purified and the enzymatic and degradation characteristics were analyzed. [Results] Maximum expression was achieved when induced by0.1 mmol/L IPTG(Isopropyl-β-D-Thiogalactoside) at 28 °C for 6 hours and the specific activity of the purified enzyme was 21 U/mg. Characterization of the enzyme showed that the optimal condition for SHA-4 includes the temperature at 37 °C, pH at 7.5, and PolyMG(heteropolymeric MG blocks) as a preferred substrate. The activity of this enzyme was inhibited by Na+ and significantly elevated to about168% by Cu~(2+). Km and Vmax of SHA-4 when used to catalyze alginate were 2.5 mg/mL and8.7 mg/(mL·min), respectively. SHA-4 was an exo-type alginate lyase with monosaccharides as the final degradation products. [Conclusion] An alginate lyase SHA-4 isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52 is successfully expressed. Among other PL6 family members, SHA-4 is the first exo-type alginate lyase with PolyMG as a preferred substrate. Along with its relatively high enzymatic activity, SHA-4 may serve as a tool enzyme with promising application and provide new clues for further exploration of the mechanism of alginate degradation.
引文
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[8]Wakabayashi M,Sakatoku A,Noda F,at el.Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles[J].Biodegradation,2012,23(1):93-105
[9]Sim SJ,Baik KS,Park SC,et al.Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone[J].Journal of Industrial Microbiology&Biotechnology,2012,39(4):585-593
[10]An QD,Zhang GL,Wu HT,et al.Properties of an alginate-degrading Flavobacterium sp.strain LXA isolated from rotting algae from coastal China[J].Canadian Journal of Microbiology,2008,54(4):314-320
[11]Kobayashi T,Uchimura K,Miyazaki M,at el.A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.[J].Extremophiles,2009,13(1):121-129
[12]Xu QQ,Zhu YX,Wang KP,at el.Cloning and identification of alginate lyases genes from Vibrio alginolyticus ATCC 17749[J].Journal of East China University of Science and Technology(Natural Science Edition),2013,39(1):29-34,41(in Chinese)胥晴晴,朱云霞,王克平,等.解藻酸弧菌株ATCC 17749中海藻酸裂解酶基因的克隆与鉴定[J].华东理工大学学报:自然科学版,2013,39(1):29-34,41
[13]Yang XM,Li SY,Wu Y,et al.Cloning and characterization of two thermo-and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp.[J].FEMS Microbiology Letters,2016,363(9):fnw079
[14]Gacesa P,Leaves GS,Weightman AJ.Genetic analysis of Klebsiella pneumoniae alginate lyase by transposon Tn10mutagenesis[J].Hydrobiologia,1987,151-152(1):571-575
[15]Hashimoto W,Miyake O,Ochiai A,et al.Molecular identification of Sphingomonas sp.A1 alginate lyase(A1-IV')as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution[J].Journal of Bioscience and Bioengineering,2005,99(1):48-54
[16]Kim HT,Chung JH,Wang DM,et al.Depolymerization of alginate into a monomeric sugar acid using Alg17C,an exo-oligoalginate lyase cloned from Saccharophagus degradans2-40[J].Applied Microbiology and Biotechnology,2012,93(5):2233-2239
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[19]Shin JW,Lee OK,Park HH,et al.Molecular characterization of a novel oligoalginate lyase consisting of AlgL-and heparinase II/III-like domains from Stenotrophomonas maltophilia KJ-2and its application to alginate saccharification[J].Korean Journal of Chemical Engineering,2015,32(5):917-924
[20]Wang LN,Li SY,Yu WG,et al.Cloning,overexpression and characterization of a new oligoalginate lyase from a marine bacterium,Shewanella sp.[J].Biotechnology Letters,2015,37(3):665-671
[21]Ochiai A,Yamasaki M,Mikami B,et al.Crystal structure of exotype alginate lyase Atu3025 from Agrobacterium tumefaciens[J].Journal of Biological Chemistry,2010,285(32):24519-24528
[22]Mori T,Takahashi M,Tanaka R,et al.Falsirhodobacter sp.alg1harbors single homologs of endo and exo-type alginate lyases efficient for alginate depolymerization[J].PLoS One,2016,11(5):e0155537
[23]Li SY,Wang LN,Han F,et al.Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp.Kz7[J].Journal of Biochemistry,2016,159(1):77-86
[24]Mathieu S,Henrissat B,Labre F,et al.Functional exploration of the polysaccharide lyase family PL6[J].PLoS One,2016,11(7):e0159415
[25]Yagi H,Fujise A,Itabashi N,et al.Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp.NAP1 isolated from brown algae[J].Bioscience,Biotechnology,and Biochemistry,2016,80(12):2338-2346
[26]Lundqvist L.Structural and interaction studies of polysaccharides by NMR spectroscopy[D].Uppsala:Doctoral Dissertation of Swedish University of Agricultural Sciences,2015
[27]Xu F,Dong F,Wang P,et al.Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGCfrom polysaccharide lyase family 6[J].Journal of Biological Chemistry,2017,292(11):4457-4468
[28]Zhu BW,Sun YD,Ni FQ,et al.Characterization of a new endo-type alginate lyase from Vibrio sp.W13[J].International Journal of Biological Macromolecules,2015,75:330-337
[29]Heyraud A,Colin-Morel P,Girond S,et al.HPLC analysis of saturated or unsaturated oligoguluronates and oligomannuronates.application to the determination of the action pattern of Haliotis tuberculata alginate lyase[J].Carbohydrate Research,1996,291:115-126
[30]Shen DX,Feng ZC,Du J.The comparison of bacterial DNAextraction methods[J].Central Plains Medical Journal,2004,31(10):20-22(in Chinese)沈德新,封志纯,杜江.细菌DNA提取方法比较[J].中原医刊,2004,31(10):20-22
[31]Ameirika,Sha HX,Hwang JS.Identification of a target protein of Hydra actinoporin-like toxin-1(HALT-1)using GST affinity purification and SILAC-based quantitative proteomics[J].Toxicon,2017,133:153-161
[32]Yoon HJ,Hashimoto W,Miyake O,et al.Overexpression in Escherichia coli,purification,and characterization of Sphingomonas sp.A1 alginate lyases[J].Protein Expression and Purification,2000,19(1):84-90
[33]Hu T,Li CX,Zhao X,et al.Preparation and characterization of guluronic acid oligosaccharides degraded by a rapid microwave irradiation method[J].Carbohydrate Research,2013,373:53-58
[34]Qian L,Tang LW,Huang SS,et al.Research progress of bioethanol from alginate fermentation[J].China Biotechnology,2013,33(1):122-127(in Chinese)钱龙,唐丽薇,黄庶识,等.海藻酸转化生物乙醇研究进展[J].中国生物工程杂志,2013,33(1):122-127
[35]Lee SI,Choi SH,Lee EY,et al.Molecular cloning,purification,and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2[J].Applied Microbiology and Biotechnology,2012,95(6):1643-1653
[36]Maki H,Mori A,Fujiyama K,et al.Cloning,sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp.OS-ALG-9[J].Journal of General Microbiology,1993,139(5):987-993
[37]Hirayama M,Hashimoto W,Murata K,et al.Comparative characterization of three bacterial exo-type alginate lyases[J].International Journal of Biological Macromolecules,2016,86:519-524
[38]Peci?a A,Pascual A,Paneque A.Cloning and Expression of the algL Gene,Encoding the Azotobacter chroococcum alginate lyase:purification and characterization of the enzyme[J].Journal of Bacteriology,1999,181(5):1409-1414
[39]Sun LP,Xue CH,Xu JC,et al.Purification and charaterization of alginate lyase from Chlorostoma rustica[J].Journal of Fishery Sciences of China,2004,11(3):266-271(in Chinese)孙丽萍,薛长湖,许家超,等.锈凹螺褐藻胶裂解酶的分离纯化及性质研究[J].中国水产科学,2004,11(3):266-271
[2]Enquist-Newman M,Faust AME,Bravo DD,et al.Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform[J].Nature,2014,505(7482):239-243
[3]Qin GK,Zhang YZ,Chen XL,at el.Research advances on alginate lyases[J].China Biotechnology,2004,24(2):26-29,33(in Chinese)秦国奎,张玉忠,陈秀兰,等.海藻酸盐裂解酶研究进展[J].中国生物工程杂志,2004,24(2):26-29,33
[4]Zhu BW,Yin H.Alginate lyase:review of major sources and classification,properties,structure-function analysis and applications[J].Bioengineered,2015,6(3):125-131
[5]Sawant SS,Salunke BK,Kim BS.A rapid,sensitive,simple plate assay for detection of microbial alginate lyase activity[J].Enzyme and Microbial Technology,2015,77:8-13
[6]Ertesv?g H,Erlien F,Skj?k-Braek G,et a1.Biochemical properties and substrate specificities of a recombinantly produced Azotobacter vinelandii alginate lyase[J].Journal of Bacteriology,1998,180(15):3779-3784
[7]Shimizu E,Ojima T,Nishita K.cDNA cloning of an alginate lyase from abalone,Haliotis discus hannai[J].Carbohydrate Research,2003,338(24):2841-2852
[8]Wakabayashi M,Sakatoku A,Noda F,at el.Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles[J].Biodegradation,2012,23(1):93-105
[9]Sim SJ,Baik KS,Park SC,et al.Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone[J].Journal of Industrial Microbiology&Biotechnology,2012,39(4):585-593
[10]An QD,Zhang GL,Wu HT,et al.Properties of an alginate-degrading Flavobacterium sp.strain LXA isolated from rotting algae from coastal China[J].Canadian Journal of Microbiology,2008,54(4):314-320
[11]Kobayashi T,Uchimura K,Miyazaki M,at el.A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp.[J].Extremophiles,2009,13(1):121-129
[12]Xu QQ,Zhu YX,Wang KP,at el.Cloning and identification of alginate lyases genes from Vibrio alginolyticus ATCC 17749[J].Journal of East China University of Science and Technology(Natural Science Edition),2013,39(1):29-34,41(in Chinese)胥晴晴,朱云霞,王克平,等.解藻酸弧菌株ATCC 17749中海藻酸裂解酶基因的克隆与鉴定[J].华东理工大学学报:自然科学版,2013,39(1):29-34,41
[13]Yang XM,Li SY,Wu Y,et al.Cloning and characterization of two thermo-and salt-tolerant oligoalginate lyases from marine bacterium Halomonas sp.[J].FEMS Microbiology Letters,2016,363(9):fnw079
[14]Gacesa P,Leaves GS,Weightman AJ.Genetic analysis of Klebsiella pneumoniae alginate lyase by transposon Tn10mutagenesis[J].Hydrobiologia,1987,151-152(1):571-575
[15]Hashimoto W,Miyake O,Ochiai A,et al.Molecular identification of Sphingomonas sp.A1 alginate lyase(A1-IV')as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution[J].Journal of Bioscience and Bioengineering,2005,99(1):48-54
[16]Kim HT,Chung JH,Wang DM,et al.Depolymerization of alginate into a monomeric sugar acid using Alg17C,an exo-oligoalginate lyase cloned from Saccharophagus degradans2-40[J].Applied Microbiology and Biotechnology,2012,93(5):2233-2239
[17]Ryu M,Lee EY.Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp.MJ-3[J].Journal of Industrial and Engineering Chemistry,2011,17(5/6):853-858
[18]Jagtap SS,Hehemann JH,Polz MF,et al.Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope,temperature,and pH adaptations[J].Applied and Environmental Microbiology,2014,80(14):4207-4214
[19]Shin JW,Lee OK,Park HH,et al.Molecular characterization of a novel oligoalginate lyase consisting of AlgL-and heparinase II/III-like domains from Stenotrophomonas maltophilia KJ-2and its application to alginate saccharification[J].Korean Journal of Chemical Engineering,2015,32(5):917-924
[20]Wang LN,Li SY,Yu WG,et al.Cloning,overexpression and characterization of a new oligoalginate lyase from a marine bacterium,Shewanella sp.[J].Biotechnology Letters,2015,37(3):665-671
[21]Ochiai A,Yamasaki M,Mikami B,et al.Crystal structure of exotype alginate lyase Atu3025 from Agrobacterium tumefaciens[J].Journal of Biological Chemistry,2010,285(32):24519-24528
[22]Mori T,Takahashi M,Tanaka R,et al.Falsirhodobacter sp.alg1harbors single homologs of endo and exo-type alginate lyases efficient for alginate depolymerization[J].PLoS One,2016,11(5):e0155537
[23]Li SY,Wang LN,Han F,et al.Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp.Kz7[J].Journal of Biochemistry,2016,159(1):77-86
[24]Mathieu S,Henrissat B,Labre F,et al.Functional exploration of the polysaccharide lyase family PL6[J].PLoS One,2016,11(7):e0159415
[25]Yagi H,Fujise A,Itabashi N,et al.Purification and characterization of a novel alginate lyase from the marine bacterium Cobetia sp.NAP1 isolated from brown algae[J].Bioscience,Biotechnology,and Biochemistry,2016,80(12):2338-2346
[26]Lundqvist L.Structural and interaction studies of polysaccharides by NMR spectroscopy[D].Uppsala:Doctoral Dissertation of Swedish University of Agricultural Sciences,2015
[27]Xu F,Dong F,Wang P,et al.Novel molecular insights into the catalytic mechanism of marine bacterial alginate lyase AlyGCfrom polysaccharide lyase family 6[J].Journal of Biological Chemistry,2017,292(11):4457-4468
[28]Zhu BW,Sun YD,Ni FQ,et al.Characterization of a new endo-type alginate lyase from Vibrio sp.W13[J].International Journal of Biological Macromolecules,2015,75:330-337
[29]Heyraud A,Colin-Morel P,Girond S,et al.HPLC analysis of saturated or unsaturated oligoguluronates and oligomannuronates.application to the determination of the action pattern of Haliotis tuberculata alginate lyase[J].Carbohydrate Research,1996,291:115-126
[30]Shen DX,Feng ZC,Du J.The comparison of bacterial DNAextraction methods[J].Central Plains Medical Journal,2004,31(10):20-22(in Chinese)沈德新,封志纯,杜江.细菌DNA提取方法比较[J].中原医刊,2004,31(10):20-22
[31]Ameirika,Sha HX,Hwang JS.Identification of a target protein of Hydra actinoporin-like toxin-1(HALT-1)using GST affinity purification and SILAC-based quantitative proteomics[J].Toxicon,2017,133:153-161
[32]Yoon HJ,Hashimoto W,Miyake O,et al.Overexpression in Escherichia coli,purification,and characterization of Sphingomonas sp.A1 alginate lyases[J].Protein Expression and Purification,2000,19(1):84-90
[33]Hu T,Li CX,Zhao X,et al.Preparation and characterization of guluronic acid oligosaccharides degraded by a rapid microwave irradiation method[J].Carbohydrate Research,2013,373:53-58
[34]Qian L,Tang LW,Huang SS,et al.Research progress of bioethanol from alginate fermentation[J].China Biotechnology,2013,33(1):122-127(in Chinese)钱龙,唐丽薇,黄庶识,等.海藻酸转化生物乙醇研究进展[J].中国生物工程杂志,2013,33(1):122-127
[35]Lee SI,Choi SH,Lee EY,et al.Molecular cloning,purification,and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2[J].Applied Microbiology and Biotechnology,2012,95(6):1643-1653
[36]Maki H,Mori A,Fujiyama K,et al.Cloning,sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp.OS-ALG-9[J].Journal of General Microbiology,1993,139(5):987-993
[37]Hirayama M,Hashimoto W,Murata K,et al.Comparative characterization of three bacterial exo-type alginate lyases[J].International Journal of Biological Macromolecules,2016,86:519-524
[38]Peci?a A,Pascual A,Paneque A.Cloning and Expression of the algL Gene,Encoding the Azotobacter chroococcum alginate lyase:purification and characterization of the enzyme[J].Journal of Bacteriology,1999,181(5):1409-1414
[39]Sun LP,Xue CH,Xu JC,et al.Purification and charaterization of alginate lyase from Chlorostoma rustica[J].Journal of Fishery Sciences of China,2004,11(3):266-271(in Chinese)孙丽萍,薛长湖,许家超,等.锈凹螺褐藻胶裂解酶的分离纯化及性质研究[J].中国水产科学,2004,11(3):266-271